CONTEXT: Appropriate glucocorticoid replacement therapy in adrenal insufficiency (AI) is crucial, given to the risks of chronic under- or over treatment, particularly in patients on multiple medications. Salivary sampling allows non-invasive, stress-free cortisol measurement. OBJECTIVE: To determine whether salivary cortisol measurement is helpful in assessing the adequacy of glucocorticoid therapy with cortisone acetate (CA) in patients with secondary AI. DESIGN: A prospective cohort study at the Endocrinology Unit of Padua University-Hospital. METHODS: Six samples of salivary cortisol were collected from 28 patients with secondary AI on CA treatment and from 36 healthy volunteers at fixed times of the day, and used to calculate salivary cortisol levels at each time-point and the Area Under Curve (AUC) across the different sampling times. RESULTS: Salivary cortisol levels were lower in patients than controls in the morning but no differences were found in the afternoon or at night before resting. Salivary cortisol levels were higher in patients immediately following CA administration. Ten patients showed an AUC above the 97.5th percentile of controls, without clinical signs of hypercortisolism, and salivary cortisol levels 90 minutes after each dose of CA predict the AUC. All patients had severe GH-deficiency: there were no differences in salivary cortisol levels or AUC between patients treated or not with GH. CONCLUSIONS: Two salivary cortisol determinations, able to predict the daily AUC, may allow assessment of the adequacy of glucocorticoid replacement therapy in secondary AI and for identifying cases of over- or under-treatment.